Mutant polymerases and uses thereof

ABSTRACT

Provided are mutant polymerases having DNA polymerase activity and reverse transcriptase activity or strand displacement activity, along with target nucleic acid amplification methods employing such mutant polymerases.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 17/303,445, filed May 28, 2021, which is a continuation of U.S.patent application Ser. No. 15/572,693, filed Nov. 8, 2017, which is theU.S. National Stage Application of PCT Application No.PCT/US2016/032041, filed May 12, 2016, which claims the benefit of U.S.Provisional Application Ser. No. 62/160,404, filed May 12, 2015. Each ofthe above-cited applications is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number1R21HG006291 awarded by National Institutes of Health; 2R44GM088948awarded by National Institutes of Health; and IIP-1127479 awarded byNational Science Foundation. The government has certain rights in theinvention.

MATERIAL INCORPORATED-BY-REFERENCE

The contents of the XML file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: P13550US03.xml, datecreated: Sep. 26, 2023, file size 43,907 bytes).

BACKGROUND OF THE INVENTION

Despite a close similarity of two well-known thermotolerant DNApolymerases (Taq large fragment, known as Klentaq1 (SEQ ID NO: 2), andBst large fragment, e.g., SEQ ID NO: 10) as to their crystal structuresand 50% sequence identity, only Taq (SEQ ID NO: 1) and Klentaq1 (SEQ IDNO: 2) DNA polymerase can catalyze PCR (during which enzyme mustrepeatedly withstand 94° C.) but the analogous Bst DNA polymerasedenatures at 65-70° C. and therefore cannot catalyze PCR. Klentaq1 (SEQID NO: 1) cannot copy double-stranded DNA by displacing a DNA strandahead of it, and the 5′-(flap) endonuclease of Taq (SEQ ID NO: 1)degrades displaced DNA; but Bst can efficiently strand-displace withoutdegradation, and hence is conventionally used to catalyze loop-mediatedisothermal amplification (LAMP), which requires double-strand invasionand efficient strand-displacement. None of these enzymes (e.g., Taq,Klentaq, Bst) are known for reverse transcriptase (RT) activity, soconventionally a separate RT enzyme is combined with them to accomplishRT-PCR (reverse transcriptase polymerase chain reaction) or likewiseRT-LAMP.

The mechanism of DNA polymerases of family A can be anthropomorphicallyunderstood as fingers and thumb, opening and closing, since theyresemble a right hand. The crystal structure of Klentaq1 (SEQ ID NO: 2)with primer/template DNA has been shown to demonstrate both open andclosed forms in the same crystal (Li et al. 1998 The EMBO Journal 17,7514-7525). Dozens of crystal structures of each of these enzymes havenot yet explained why Klentaq1 enzyme cannot perform the functionsexhibited by other DNA polymerases, such as copying RNA into DNA (i.e.,reverse transcriptase activity) or copying double-stranded DNA(displacing the non-template strand, or strand displacement activity),which can be catalyzed by the homologous large fragment of Bst DNApolymerase (e.g., SEQ ID NO: 10 or Bst 2.0, New England Biolabs), usedfor the isothermal method LAMP (Tomita et al. 2008 Nature Protocols 3,877-882). Also unexplained are the mechanisms of (mostly unknown)inhibitor chemicals that can prevent successful PCR analysis of blood,urine or food-safety cultures. Food safety assays, often even afterpathogen enrichment culture and partial DNA purification, are unreliabledue to inhibitors, particularly for chocolate and black pepper. Foodsafety analysis can be limited for PCR and RT-PCR by enzyme inhibitors,particularly chocolate and pepper. As such, food or food-bacteriaenriched culture RNA or DNA may require purification before PCRanalysis, with varying success, convenience, and expense.

Although full-length, wild-type Taq (SEQ ID NO: 1) can strand-displaceto some extent, its 5′-endonuclease (flap endonuclease, FEN), accordingto previous studies using only enzymes without this domain, cleaves thedisplaced strand so much that it is not appropriate for LAMP. Twostudies have selected different combinations of four amino acid changesthat demonstrate reverse transcriptase activity for Klentaq1 (Blatter etal. 2013 Angewandte Chemie International Edition 52, 11935-11939) or TaqDNA polymerase (Ong et al. 2006 Journal of Molecular Biology 361,537-550).

Known mutant polymerases include Omni Taq, i.e., FL-22 (as described inU.S. Patent Application Publication No. 2011/0027832) and Omni Klentaq,i.e., KlenTaq-10 (as described in U.S. Patent Application PublicationNo. 2006/0084074). Known mutant polymerases and uses thereof aredescribed in, for example, U.S. Pat. No. 7,462,475, issued 9 Dec. 2008;U.S. Patent Application Publication No. 2009/0170060, published 2 Jul.2009; U.S. Patent Application Publication No. 2011/0027832, published 3Feb. 2011; U.S. Patent Application Publication No. 2012/0028259,published 2 Feb. 2012; international PCT application WO2012/088479,published 28 Jun. 2012; U.S. Patent Application Publication No.2014/0113299, published 24 Apr. 2014.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofmutant polymerases having DNA polymerase activity and reversetranscriptase activity or strand displacement activity.

Another aspect provides for methods of amplifying a target nucleic acidusing mutant polymerases having DNA polymerase activity and reversetranscriptase activity or strand displacement activity.

In some embodiments of the methods, amplifying comprises reversetranscriptase PCR (RT-PCR). In some embodiments, amplifying comprisesloop-mediated isothermal amplification (LAMP). In some embodiments, LAMPis performed with a hanging drop hot start. In some embodiments,amplifying comprises reverse transcriptase loop-mediated isothermalamplification (RT-LAMP). In some embodiments, RT-LAMP is performed witha hanging drop hot start.

In some embodiments of the methods, the mutant polymerase includes apolypeptide having (a) at least 95% sequence identity to SEQ ID NO: 1 orSEQ ID NO: 2, (b) DNA polymerase activity and reverse transcriptaseactivity and/or strand displacement activity, (c) at least onesubstitution selected from the group consisting of D732N and thecombination of E742R and A743R (EA742RR) (per wild type Taq numbering),and (iv) optionally, a F1AsH insertion, such as FLNCCPGCC betweenposition 738 and position 739 (per wild type Taq numbering). In someembodiments, the mutant polymerase further includes at least onemutation selected from the group consisting of L609P, E626K, V649I,I707L, E708K, E708L, E708N, E708Q, E708I, E708W, E708R, E708V, E708S,E404G, G418E, V453L, A454S, R487G, I528M, L533R, D551G, D578E, I599V,L657Q, K738R, L781 I, and E818V (per wild-type Taq numbering), or anycombination thereof. In other embodiments, the mutant polymerase furtherincludes at least one mutation selected from the group consisting ofD119A, D119N, E742R and A743R (per wild-type Taq numbering), or anycombination thereof.

In some embodiments of the methods, the amplifying comprises an assaymixture including (A) a sample comprising a target RNA, (B) primersspecific for the target RNA or cDNA transcribed from the target RNA, (C)a buffer, and (D) at least one mutant polymerase comprising apolypeptide sequence having (i) at least 95% sequence identity to SEQ IDNO: 1 or SEQ ID NO: 2, (ii) reverse transcriptase activity and DNApolymerase activity, and (iii) at least one substitution selected fromthe group consisting of D732N and the combination of E742R and A743R(EA742RR) (per wild type Taq numbering); and amplifying the targetnucleic acid in the assay mixture in RT-PCR.

In some embodiments of the methods, the assay mixture does not include aseparate reverse transcription enzyme or Mn⁺⁺ ion.

In some embodiments of the methods, the assay mixture includes a sampleincluding a target RNA not purified prior to addition to the assaymixture.

In some embodiments of the methods, the assay mixture includes aninhibitory substance in an amount sufficient to cause a wild type Taqpolymerase to fail to amplify the target nucleic acid in the RT-PCR. Incertain embodiments of the methods, the inhibitory substance includeswhole blood, a blood traction, chocolate, peanut butter, milk, seafood,meat, egg, or a soil extract.

In some embodiments of the methods, the PCR is a real-time PCR; theassay mixture further comprises at least one dye; and amplifying thetarget nucleic acid comprises amplifying the target nucleic acid in theassay mixture in a real-time PCR.

In some embodiments of the methods, the mutant polymerase includes atleast one mutant polymerase including a F1AsH insertion of FLNCCPGCCbetween position 738 and position 739 (per wild type Taq numbering).

Another aspect provides for a kit for use of methods of amplifying atarget nucleic acid using mutant polymerases having DNA polymeraseactivity and reverse transcriptase activity or strand displacementactivity.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 is a diagram showing phenotypes of a variety of polymerases. Thehot spring DNA polymerase called Bst large fragment, Bst 2.0, isavailable commercially (“Manta”, NE Biolabs). While Bst large fragmentis very similar to Klentaq1, it cannot stand more than 65° C. (70° C.for Bst 2.0) so it cannot do PCR, which requires thermostability to 95°C. The crystal structures of Klentaq1 and Bst large fragment are verysimilar, and they are 50% homologous by amino acid sequence. Full-lengthTaq has an N-terminal domain that cuts displaced DNA (“flaps”) ahead ofit.

FIG. 2 shows a crystal structure of Klentaq1. Incoming DNA templatetakes a sharp turn as it enters the enzyme to be copied (shownapproximately by the dotted line). This is more clearly visible on thecrystal structure of Bst DNA polymerase. Numbering is from wild-type,full-length Taq. Amino acids 742,743 are adjacent to the templatestrand, and several mutations here have been described (see e.g.,Yamagami et al. 2014 Front Microbiol 5:461). But amino acid 732 is notnear DNA in the crystal structure. Also indicated are indicated aminoacids 738,739, where inserts can be placed while retaining activity.Other insert positions are disclosed in Yamagami et al. 2014 FrontMicrobiol 5:461.

FIG. 3 is a diagram showing F1AsH, a label that attaches only to CCXXCCat the double cysteines (see e.g., Tsien 2005 Nat Biotechnol 23,1308-1314). A preferred sequence can be FLNCCPGCCMEP. Several versionsof F1AsH were inserted between 738,739 (per wild type Taq numbering) ofKlentaq1, as further described herein.

FIG. 4 is a diagram illustrating loop-mediated isothermal amplification(LAMP). In short, LAMP primers (e.g., from a set of six) “FiP” and “BiP”(or rather their complements after they are copied) hook back onthemselves and prime synthesis the other way, at isothermal temperaturesof about 60-70° C. LAMP is further described herein.

FIG. 5 shows real time PCR results from a cracked pepper screening assayfor resistance using whole E. coli as PCR enzyme. Presented is raw dataof 96 curves of SyBr Green fluorescence. Rising curves from cycle 20-40represent successful PC or 4 less-resistant controls. Five ill of waterextract of black pepper was included in each reaction. The highestrising curve was Taq DNA polymerase clone A111 (SEQ ID NO: 3), with asingle mutation D732N. Resistance to chocolate, blood and bile wassubsequently observed for enzyme A111.

FIG. 6 shows a strand-displacement half-dumbbell test. This assay testedwhether an enzyme could displace 1.2 kb, go around a loop, and come backto an end, producing a predicted, discrete 2.4 kb band. FIG. 6 alsoshows a cartoon of the 1.2 kb loop structure and the 2.4 kb displacedstrand. This assay was designed so that strand-displacement would causea band at 2.4 kb, rather than a smear with other assays. Mutant A111,i.e., TaqD732N (SEQ ID NO: 3) did surprisingly well in this assay.

FIG. 7 shows four-primer PCR as a test to compare mutant polymerases. Itwas thought that Klentaq1 (SEQ ID NO: 2) could PCR-amplify only theinner-primed product, since it cannot start at the outer-nested primersand displace the inner primed DNA that is already there. D732N can dosome larger product. The largest expected product is not made by any ofthe enzymes depicted in FIG. 7 .

FIG. 8 is a gel image showing a four-primer PCR test. Wild-type Taq(lane 1) actually can make all four expected products. FIG. 8 shows thatwhen the F1AsH-binding sequence was inserted between positions 738 and739, and the polymerase also contained the D732N, E742R, and A743Rmutations, differences in strand displacement efficiency were observeddepending on the temperature of the PCR extension step. ConventionalLAMP protocols are reported to need optimizing for temperature in thiszone.

FIG. 9 shows a 4 primer (non-optimized assay) for strand displacementactivity run at 60° C. and 70° C. Control was Manta polymerase (a BstDNA large fragment polymerase). Mutant polymerase KTflnC4RR (truncatedpolymerase having D732N; FLNCCPGCC insert at 738,739; and E742R andA743R (EA742RR); SEQ ID NO: 9) showed strand displacement activity,notably at 70° C. where Manta was inactivated. It was observed that LAMPwas catalyzed by a Taq or Klentaq mutant enzyme, compared to Bst DNApolymerase Manta (supplied by Enzymatics).

FIG. 10 is a diagram showing known mutations for RT activity in Taq andKlentaq (Blatter et al. 2013 Angewandte Chemie International Edition 52,11935-11939) or Taq DNA polymerase (Ong et al. 2006 Journal of MolecularBiology 361, 537-550). When the four known mutations of Blatter et al.(S515R, I638F, M747K, L459M per wild-type Taq numbering) were added toKlentaq1 having D732N, no PCR activity was observed.

FIG. 11 is an image of a LAMP assay using the 6-primer LAMP setpublished by Lucigen for RT-LAMP of MS2 RNA. Surprisingly andunexpectedly, Klentaq D732N (SEQ ID NO: 4) worked from the RNA template(see lane 2). In the 45 minute assay, mutants having both E742R andA743R substitutions (EA742RR) and F1AsH inserts at 738,739 had improvedperformance over other enzymes.

FIG. 12 is a diagram depicting hanging drop hot start for RT-PCR orRT-LAMP. No-template amplification can be caused by PCR or LAMP productcontamination in an environment. DNAse I and off cite preparation ofprimers can at least in part address such contamination. The hangingdrop hot start protocol can also be used to get RNA-dependentamplification. RNase I reduces or eliminates the RNA-amplified product(data not shown) if the RNAse I digest is done in 1/10 strength buffer.

FIG. 13 shows an image from a LAMP reaction monitored via real-timefluorescence change (using Eva Green dye) for FT732N (SEQ ID NO: 4),FT732N (SEQ ID NO: 4) preheated, and KT732-FLNCY-E742R-A743R(KT732-FLNCY-EA742RR; SEQ ID NO: 9).

FIG. 14A shows RT-LAMP real-time traces with no RT enzyme and noMn^(+/+) using Eva Green indicator for tested mutant polymerases (KT-RR,SEQ ID NO: 6; Taq-D732N, SEQ ID NO: 3; KT-732, SEQ ID NO: 4; KT-NRR, SEQID NO: 8; and KT-flnC4, SEQ ID NO: 9; Taq, SEQ ID NO: 1; TaqRR, SEQ IDNO: 5; and Klentaq1, SEQ ID NO: 2). Buffer conditions are 50 mMTris-HCl, pH 8.55, 8 mM ammonium sulfate, 200 μM each dNTP, M betaine,0.025% Brij58. On the trace diagrams, inflected curves indicate positiveLAMP reactions. FIG. 14B shows melting curves, where genuine LAMPproducts melted at 85-86.5° C. After eliminating inserts at 738,739 andthe D732N, a Klentaq1 polymerase with only E742R and A743R (EA742RR)(SEQ ID NO: 6) had the best performance of the enzymes tested. Mutantpolymerase A111 (having D732N, SEQ ID NO: 3) had surprisingly goodperformance (despite having a 5′-flap endonuclease that shouldtheoretically be preventing any LAMP) with a good curve (much lowerbackground of “primer-dimer” synthesis). Taq with E742R and A743R(EA742RR) (SEQ ID NO: 5) and Taq (SEQ ID NO: 1) did not work in theassay shown in FIG. 14 . On a gel, the Taq products look different,which may be some 5′-exonuclease activity showing.

FIG. 15 shows a gel image, amplification curves and melt curves for anMS2 RNA RT-LAMP assay for KT-DRR (SEQ ID NO: 8), NTC KT-DRR, Taq-D732N(A111, SEQ ID NO: 3), and NTC.

FIG. 16A-B shows amplification curves, melting curves and a diagram oftemperature gradient for a RT-PCR assay. Results suggest that adding anRT step would be beneficial to provide some reaction time before the PCRstarts. Results also showed improvements with “wiggle” of thetemperature of this RT step.

FIG. 17 shows ability of wild-type Taq and Taq mutant D732N to catalyzePCR reactions in the presence of various amount of chocolate.

FIG. 18 shows the ability of wild-type Taq and Taq mutant D732N tocatalyze PCR reactions in the presence of various amount of black pepperor whole blood.

FIG. 19 shows the results of RT-LAMP with full-length Taq-D732N and5′-exo mutations D119A and D119N.

FIG. 20 shows the results of RT-PCR performed with Taq mutant D732Ncompared to wild-type Taq.

FIG. 21 shows that Bst DNA polymerase, without any RT (reversetranscriptase) enzyme, can catalyze RT-LAMP using the conditionsdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thata single mutation (D732N per wild type Taq numbering) of the PCR enzymeKlentaq1 DNA polymerase can broaden its ability to include multipleactivities including reverse transcriptase (RT) activity orstrand-displacement activity. As shown herein, a mutant Klentaq1 DNApolymerase having mutation D732N and RT and strand-displacement activitycan catalyze reverse transcription loop-mediated isothermalamplification (RT-LAMP) using RNA template.

While a mutant polymerase having a D732N mutation has been associatedwith resistance to cracked pepper, chocolate, or blood (see e.g., U.S.Patent Application Publication No. 2014/0113299), the present disclosureis the first description of the surprising and unexpected RT andstrand-displacement activity.

The present disclosure is based, at least in part, on a furtherdiscovery that mutations E742R and A743R (i.e., EA742RR) (optionally incombination with D732N) can reduce the amount of time to produce atypical DNA ladder from LAMP. As shown herein, while it can take 80-100minutes using a polymerase having a D732N mutation at a constant 68° C.to produce the typical DNA ladder from LAMP with 1-2 ng of MS2 RNAtemplate, a mutant polymerase having a D732N mutation and both E742R andA743R (EA742RR) mutations can reduce this time to 30-40 minutes.

While the combined E742R and A743R (EA742RR) polymerase mutation hasbeen previously described and associated with showing higher DNAaffinity or faster primer extension ability (see e.g., Yamagami et al.2014 Front Microbiol 5:461), the present disclosure is the firstdescription of the combination of the E742R, A743R, and D732N mutationsand the surprising and unexpected RT and strand-displacement activity.Furthermore, Yamagami et al. (2014 Front Microbiol 5:461) described thecombination of E742R and A743R (EA742RR) as “too tight” for useful PCR,thereby discouraging one of ordinary skill from using this mutation in apolymerase for PCR. But as disclosed herein, for strand-displacementactivity, “tight” may be beneficial.

The present disclosure is based, at least in part, on a furtherdiscovery that mutations D119A or D119N (optionally in combination withD732N) can increase the clarity of the banding patterns for LAMP. Asshown herein, a mutant polymerase having a D732N mutation and D119A or amutant polymerase having a D732N mutation and D119N showed improvedclarity (see e.g., FIG. 19 ).

As a further modification, a mutant polymerase having a D732N mutation;D732N, E742R, and A743R; D732N and D119A; or D732N and D119N; mutationscan be combined with an insertion to allow fluorescent labeling withdifluoro-F1AsH near the path of the DNA template. As shown herein,D732N, E742R, and A743R mutations were combined with an insertionFLNCCPGCC at 738,739 in a mutant polymerase to allow fluorescentlabeling with difluoro-F1AsH near the path of the DNA template, forpotential observation of single molecules of enzyme in action for a DNAsequencing method. As shown herein, without the F1AsH attachment, themutant enzyme can still do PCR and RT-LAMP; in some embodiments,apparent activity can be reduced but still evident when the F1AsH isadded.

The present disclosure is based, at least in part, on a furtherdiscovery of a method of RT-LAMP reliably dependent on the addition ofMS2 RNA. It has been reported that performance of RT-LAMP can result incontamination of a surrounding environment with DNA product to the pointthat template is sometimes not required, or template RNA only enhancesthe appearance of product. Conventional solutions to this problem use 5dNTPs G, A, C, T, and U coupled with eventual use of uracildeglycosylase.

A “hanging hot start” method using DNase I as disclosed can overcomereported RT-LAMP contamination issues. As shown herein, primers can beprepared and diluted in a non-contaminated environment, and added (e.g.,in about 2-4 μl) to the cap of each reaction tube. The rest of thereaction (buffer, DNA polymerase, and MS2 RNA, etc.) can be set up(e.g., in about 23-25 μl) at the bottom of the reaction tube withRNase-free DNase I and CaCl₂ (e.g., about 0.1 mM) for a period of time(e.g., about 10-30 minutes) at a temperature (e.g., room temperature).The reaction tubes can be gently moved to a thermal cycler (which is notto be cycled) with the primers still hanging on the cap. After the blockhas warmed (e.g., to 68° C. or 75° C.) for a period of time (e.g., about1 minute), the DNase I is inactive, and warm tubes can be removed fromthe hot block and mixed (e.g., flicked sharply downward to dislodge theprimers into the reactions), then can be returned (e.g., returnedimmediately) to the hot block for incubation at a set temperature (e.g.,68° C.). With the above described protocol, RT-LAMP reactions can bereliably dependent on the addition of MS2 RNA, thereby overcoming inwhole or in part the recognized contamination issues.

The following U.S. patent applications are incorporated herein byreference in their entirety: U.S. Pat. No. 7,462,475, issued 9 Dec.2008; U.S. Patent Application Publication No. 2009/0170060, published 2Jul. 2009; U.S. Patent Application Publication No. 2011/0027832,published 3 Feb. 2011; U.S. Patent Application Publication No.2012/0028259, published 2 Feb. 2012; international PCT applicationWO2012/088479, published 28 Jun. 2012; and U.S. Patent ApplicationPublication No. 2014/0113299, published 24 Apr. 2014. Except asotherwise noted herein, therefore, the process of the present disclosurecan be carried out in accordance with compositions or processes of thesereferences.

Mutant Polymerases

Some embodiments provide mutant polymerases having polymerase activity,reverse transcriptase activity (RT), or strand-displacement activity.For example, some mutant polymerases described herein have polymeraseactivity, RT activity, and strand-displacement activity. Such mutantpolymerases can be used in, e.g., RT-PCR, LAMP PCR, or RT-LAMP PCR.

According to conventional notation, amino acid mutations discussedherein may be represented, from left to right, by the one letter codefor the wild type amino acid, the amino acid position number, and theone letter code for the mutant amino acid. For mutant polypeptidesequences, an amino acid different than corresponding wild type may berepresented, from left to right, by the amino acid position number andthe one letter code for the amino acid that is different thancorresponding wild type.

A “variant” polypeptide described in the following paragraphs is asdefined in the “variant” section further below. Exemplary sequenceidentity (e.g., at least about 95% sequence identity) is not meant tolimit the full range of sequence identity as discussed in the “variant”section herein.

For the following discussion, wild type Taq numbering (corresponding tonumbering of full-length Taq of SEQ ID NO: 1) is used in thisdescriptive text so as to make clear the relationship between thepolypeptides. Wild type Taq (SEQ ID NO: 1) and truncated Klentaq-1 (SEQID NO: 2) have complete sequence homology across positions 279-832 ofSEQ ID NO: 1, except for positions 279 (Gly) and 280 (Ser) of SEQ ID NO:1 (corresponding to positions 1 (Met) and 2 (Gly) of truncated SEQ IDNO: 2). Klentaq-1 (SEQ ID NO: 2) has an open reading frame sequencestarting with codons for amino acids methionine (M), glycine (G),leucine (L), leucine (L), histidine (H), glutamic acid (E), phenylanine(F). The amino acid changes at 279-280 of wild type Taq (SEQ ID NO: 1)and positions 1-2 of truncated Klentaq-1 (SEQ ID NO: 2) are notnecessarily associated with a difference in phenotype as describedherein.

With respect to wild-type Taq numbering, for truncated polymerasepolypeptides (e.g., Klentaq-1 of SEQ ID NO: 2), position number 1 asnotated in the Sequence Listing of SEQ ID NO: 2 corresponds to positionnumber 279 as notated in the full-length Taq of SEQ ID NO: 1. Similarly,position number 2 of SEQ ID NO: 2 corresponds to position number 280 ofSEQ ID NO: 1. Similarly, position number 454 of SEQ ID NO: 2 correspondsto position number 732 of SEQ ID NO: 1. Similarly, position number 464of SEQ ID NO: 2 corresponds to position number 742 of SEQ ID NO: 1.Similarly, position number 465 of SEQ ID NO: 2 corresponds to positionnumber 743 of SEQ ID NO: 1. In other words, one can determine thecorresponding position in full-length SEQ ID NO: 1 by adding 278 the anyposition in SEQ ID NO: 2.

A mutant polymerase described herein can be produced according tomethods known in the art. For example, oligonucleotides providing thespecific amino acid changes to a mutant polymerase described can beprepared by standard synthetic techniques (e.g., an automated DNAsynthesizer) and used as PCR primers in site-directed mutagenesis.Standard procedures of expression of mutant polymerase polypeptides fromencoding DNA sequences can then be performed. Alternatively, the mutantDNA polymerase polypeptides can be directly synthesized according tomethods known in the art.

A mutant polymerase having a mutation described herein can be a fulllength mutant polymerase or a truncated mutant polymerase, as comparedto a wild-type Taq polymerase. For example, a truncated mutantpolymerase can be truncated at position 278 per wild-type Taq numbering(e.g., position 1 of the truncated mutant corresponds to position 279 ofSEQ ID NO: 1). One of skill in the art will understand that a truncatedmutant polymerase can be truncated at any position of a full lengthsequence so long as polymerase activity (or other required phenotypes,such as RT activity or strand-displacement activity) is retained.

A truncated mutant polymerase can be referred to as a “functionalfragment” of a longer polymerase, such as a full-length polymerase. Forexample, SEQ ID NO: 2 (Klentaq-1, KT-1) is a variant (having G279M andS280G per wild type Taq numbering) and functional fragment of SEQ ID NO:1 (wild type Taq). A functional fragment is shorter than the length of areference polymerase and retains polymerase activity (or other requiredphenotypes, such as RT activity or strand-displacement activity).

As disclosed herein, one or more amino acid mutations (e.g., addition,deletion, substitution) can be associated with a phenotype describedherein. In some embodiments, a mutant polymerase (e.g., a full lengthmutant polymerase or a truncated mutant polymerase) can include one ormore of mutations D732N, E742R, or A743R. For example, a mutantpolymerase (e.g., a full length mutant polymerase or a truncated mutantpolymerase) can include mutation D732N. As another example, a mutantpolymerase (e.g., a full length mutant polymerase or a truncated mutantpolymerase) can include mutations E742R and A743R. As another example, amutant polymerase (e.g., a full length mutant polymerase or a truncatedmutant polymerase) can include mutations D732N, E742R, and A743R. Thecombination of E742R and A743R can also be referred to herein asEA742RR.

For example, a mutant polymerase can include an amino acid sequence ofSEQ ID NO: 1 having a D732N substitution, or a variant (e.g., at leastabout 95% sequence identity) thereof having at least the D732Nsubstitution and having polymerase activity and RT activity or stranddisplacement activity. A full length mutant polymerase having a D732Nsubstitution can be mutant polymerase A-111 as identified in U.S. PatentApplication Publication No. 2014/0113299. As demonstrated, full lengthA-111 having D732N (SEQ ID NO: 3) can overcome enzyme inhibitors inchocolate, pepper and cheese, thereby reducing or eliminatingpurification of food or food-bacteria culture RNA or DNA.

Full length A-111 having D732N (SEQ ID NO: 3) can show smearing on anagarose gel when tested for normal PCR with longer products. This may bedue to higher than normal template-switching, in which astrand-displacing enzyme would be expected to be good at. Full lengthA-111 having D732N (SEQ ID NO: 3) can reduce maximum yield of PCRproduct with a reduced amount of input template.

As another example, a mutant polymerase can include an amino acidsequence of SEQ ID NO: 2 having a D732N mutation (per wild-type Taqnumbering), or a variant (e.g., at least about 95% sequence identity)thereof having at least the D732N substitution and having polymeraseactivity and RT activity or strand displacement activity. Note that D454in SEQ ID NO: 2 corresponds to D732 according to wild type Taqnumbering.

As another example, a mutant polymerase can include an amino acidsequence of SEQ ID NO: 1 having a E742R substitution and a A743Rsubstitution (which can be referred to as EA742RR), or a variant (e.g.,at least about 95% sequence identity) thereof having at least the E742Rand A743R substitutions and having polymerase activity and RT activityor strand displacement activity.

As another example, a mutant polymerase can include an amino acidsequence of SEQ ID NO: 2 having an E742R substitution and an A743Rsubstitution (per wild-type Taq numbering), or a variant (e.g., at leastabout 95% sequence identity) thereof having at least the E742R and A743Rsubstitutions and having polymerase activity and RT activity or stranddisplacement activity. Note that E464 of SEQ ID NO: 2 corresponds toE742 according to wild type Taq numbering; and A465 of SEQ ID NO: 2corresponds to A743 according to wild type Taq numbering. As shownherein, a Klentaq1 double mutant having E742R and A743R substitutions(EA742RR) can requires the use of less enzyme, provides faster results,and functions in the presence of inhibitors.

As another example, a mutant polymerase can include an amino acidsequence of SEQ ID NO: 1 having a D732N substitution, a E742Rsubstitution, and a A743R substitution, or a variant (e.g., at leastabout 95% sequence identity) thereof having at least the D732N, E742R,and A743R substitutions and having polymerase activity and RT activityor strand displacement activity.

As another example, a mutant polymerase can include an amino acidsequence of SEQ ID NO: 2 having a D732N substitution, a E742Rsubstitution, and a A743R substitution (per wild-type Taq numbering), ora variant (e.g., at least about 95% sequence identity) thereof having atleast the D732N, E742R, and A743R substitutions and having polymeraseactivity and RT activity or strand displacement activity. Note that D454in SEQ ID NO: 2 corresponds to D732 according to wild type Taqnumbering; E464 of SEQ ID NO: 2 corresponds to E742 according to wildtype Taq numbering; and A465 of SEQ ID NO: 2 corresponds to A743according to wild type Taq numbering.

A mutant polymerase described herein can further include a polymerasemutation disclosed in U.S. Pat. Nos. 6,403,341; 7,393,635; 7,462,475; WO2012/088479 (and corresponding U.S. application Ser. No. 13/997,194); USPat App Pub No. 2010/0013291; US Pat App Pub No. 2012/0028259, and USPat App Pub No. 2014/0113299, each incorporated herein by reference.

In some embodiments, a mutant polymerase (e.g., a full length mutantpolymerase or a truncated mutant polymerase) can include one or more ofthe following substitutions: L609P, E626K, V649I, I707L, E708K, E708L,E708N, E708Q, E708I, E708W, E708R, E708V, E708S, E404G, G418E, V453L,A454S, R487G, I528ML533R, D551G, D578E, I599V, L657Q, K738R, L781 I, orE818V (per wild type numbering). A substitution at one or more of thesepositions (e.g., 708) can occur in combination with one or more othersubstitutions described herein. For example, a mutant polymerase (e.g.,a full length mutant polymerase or a truncated mutant polymerase) canhave (A) one or more substitutions selected from D732N, E742R, and A743Rand (B) at least one substitution selected from L609P, E626K, V649I,I707L, E708K, E708L, E708N, E708Q, E708I, E708W, E708R, E708V, E708S,E404G, G418E, V453L, A454S, R487G, I528ML533R, D551G, D578E, I599V,L657Q, K738R, L781 I, and E818V (per wild type numbering). As anotherexample, a mutant polymerase can include SEQ ID NO: 1 having (A) one ormore substitutions selected from D732N, E742R, and A743R and (B) atleast one substitution selected from L609P, E626K, V649I, I707L, E708K,E708L, E708N, E708Q, E708I, E708W, E708R, E708V, E708S, E404G, G418E,V453L, A454S, R487G, I528ML533R, D551G, D578E, I599V, L657Q, K738R,L781I, and E818V (per wild type numbering). As another example, a mutantpolymerase can include SEQ ID NO: 2 having (A) one or more substitutionsselected from D732N, E742R, and A743R (per wild-type Taq numbering) and(B) at least one substitution selected from L609P, E626K, V649I, I707L,E708K, E708L, E708N, E708Q, E708I, E708W, E708R, E708V, E708S, E404G,G418E, V453L, A454S, R487G, I528ML533R, D551G, D578E, I599V, L657Q,K738R, L781 I, and E818V (per wild-type Taq numbering).

A mutant polymerase described herein can be used in conjunction withcompositions or processes described in U.S. Pat. Nos. 6,403,341;7,393,635; 7,462,475; WO 2012/088479 (and corresponding U.S. applicationSer. No. 13/997,194); US Pat App Pub No. 2010/0013291; US Pat App PubNo. 2012/0028259, and US Pat App Pub No. 2014/0113299, each incorporatedherein by reference.

Another aspect of the present disclosure provides a polynucleotideencoding a mutant polymerase described herein. Also provided is anucleic acid construct (e.g., an expression vector) includingpolynucleotide encoding a mutant polymerase described herein. Aconstruct (e.g., a DNA construct) can include the following operablyassociated components: a promoter functional in a host cell, anucleotide sequence (e.g., a heterologous DNA sequence, an exogenous DNAsegment, or a heterologous nucleic acid) encoding a mutant polymerasedescribed herein, a transcriptional termination sequence. Generation ofan encoding polynucleotide, a nucleic acid construct (e.g., anexpression vector), transformation of a host cell with such construct,and expression of a mutant polymerase from a transformed host cell iswithin the state of the art.

Variants

The term “variant” polypeptides (or encoding polynucleotides) isdiscussed below. The description of “variant” below is incorporated byreference into each recitation of “variant” in the description of mutantpolymerases herein. For example, the full range of sequence identitydiscussed below applies equally to “variant” polypeptides discussedelsewhere herein.

Included in the scope of the present disclosure are variant polypeptides(or encoding polynucleotides) with at least 80% sequence identity tosequences described herein, so long as such variants retain (A) apolymerase activity and (B) RT activity or strand displacement activity.

For example, a variant polypeptide (or an encoding polynucleotide) withpolymerase activity can have at least about 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequenceidentity to sequences disclosed herein (including disclosed sequenceshaving substitutions described herein). It is understood that in someembodiments, “about” modifies each of these recited sequence identityvalues. A variant polypeptide (or encoding polynucleotides) withpolymerase activity, RT activity, or strand displacement activity canhave at least 95% sequence identity to a sequence disclosed herein. Avariant polypeptide (or an encoding polynucleotide) with polymeraseactivity, RT activity, or strand displacement activity can have at least99% sequence identity to a sequence disclosed herein. The species arerepresentative of the genus of variant polypeptides of each of theserespective sequences because all variants must possess the specifiedcatalytic activity (e.g., polymerase activity, RT activity, or stranddisplacement activity) and must have the percent identity required aboveto the reference sequence.

Design, generation, and testing of the variant polypeptides having theabove required percent identities to the sequences of the mutant DNApolymerases and retaining a required resistant phenotype is within theskill of the art. For example, directed evolution and rapid isolation ofmutants can be according to methods described in references including,but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688;Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) ProcNatl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art couldgenerate a large number of polypeptide variants having, for example, atleast 95-99% identity to the sequences of mutant DNA polymerasesdescribed herein and screen such for phenotypes including,dye-resistance, blood-resistance, or soil-resistance according tomethods routine in the art. Generally, conservative substitutions can bemade at any position so long as the required activity is retained.

Amino acid sequence identity percent (%) is understood as the percentageof amino acid residues that are identical with amino acid residues in acandidate sequence in comparison to a reference sequence when the twosequences are aligned. To determine percent amino acid identity,sequences are aligned and if necessary, gaps are introduced to achievethe maximum percent sequence identity; conservative substitutions arenot considered as part of the sequence identity. Amino acid sequencealignment procedures to determine percent identity are well known tothose of skill in the art. Often publicly available computer software,such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software, is used toalign peptide sequences. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared. When amino acid sequences are aligned, thepercent amino acid sequence identity of a given amino acid sequence Ato, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain percent amino acid sequence identity to, with, oragainst a given amino acid sequence B) can be calculated as: percentamino acid sequence identity=X/Y100, where X is the number of amino acidresidues scored as identical matches by the sequence alignment program'sor algorithm's alignment of A and B, and Y is the total number of aminoacid residues in B. If the length of amino acid sequence A is not equalto the length of amino acid sequence B, the percent amino acid sequenceidentity of A to B will not equal the percent amino acid sequenceidentity of B to A.

Phenotype

As described herein, a mutant polymerase described herein can havepolymerase activity, RT activity, or strand displacement activity. Asdescribed herein, a mutant polymerase can be multi-functional. Forexample, a mutant polymerase can be bifunctional. Bifunctionality caninclude polymerase activity and either RT activity or stranddisplacement activity, and therefore have PCR and LAMP functionality.

Polymerase Activity.

A polymerase enzyme is understood to a add a free nucleotide to an —OHgroup on the 3′ end of a newly forming nucleic acid strand, resulting inelongation of the strand in a 5′-3′ direction. Directionality of thenewly forming strand (the daughter strand) is understood to be oppositeto the direction in which a polymerase moves along a template strand.Thus, a polymerase moves along the template strand in a 3′-5′ direction,and the daughter strand is formed in a 5′-3′ direction. In someembodiments, polymerase activity includes the ability of a polymerase tofully or partially complete a PCR. PCR is described further below.

In some embodiments, a polymerase activity is a resistant polymeraseactivity, where the mutant polymerase is resistant to one or moresubstances that can inhibit PCR. A mutant polymerase described hereincan have a phenotype including polymerase activity and an ability tofully or partially complete a PCR in a reaction mixture including aninhibitory substance at a concentration that a control polymerase (e.g.,wild type Taq polymerase of SEQ ID NO: 1, or Klentaq1 polymerase of SEQID NO: 2) would fail to amplify a target nucleic acid. Resistantpolymerase activity can be retention of all or most polymerase activity,or sufficient polymerase to complete a PCR, in the presence of a samplecontaining one or more of chocolate, pepper, milk, seafood, meat, egg,blood, urine, humic acid, bile, or plant material in sufficient quantityto inhibit or substantially inhibit a corresponding wild typepolymerase. A mutant polymerase described herein can have a phenotypeincluding polymerase activity and an ability to fully or partiallycomplete a PCR in a reaction mixture including an inhibitory substanceat a concentration that a control (e.g., wild type Taq polymerase of SEQID NO: 1 or Klentaq1 of SEQ ID NO: 2) would fail to amplify a targetnucleic acid. An inhibitory substance can be present in food or foodsamples, such as chocolate, peanut butter, milk, seafood, meat, or egg,or other foods or food samples. GITC (guanidinium) or ethanol areexemplary inhibitory substance that can be present in an assay mixture.An inhibitory substance can be present in chocolate, pepper, blood,urine, humic acid, bile, tannins, melanin, indigo dyes, or plantmaterial. For example, an inhibitory substance can be a polyphenol, suchas a polyphenol present in a sample described above. Thus, a mutantpolymerase described herein can be used to amplify a targetpolynucleotide in a PCR in the presence of one or more inhibitorysubstances.

Generally, a mutant polymerase having a resistant polymerase activitydescribed herein can tolerate at least an order of magnitude greaterconcentration of an inhibitory substance described herein as compared toa control (e.g., wild type Taq polymerase of SEQ ID NO: 1 or Klentaq1 ofSEQ ID NO: 2). A mutant polymerase described herein can provide foramplification of a target nucleic acid in a sample containing aninhibitory substance at a level inhibitory to a wild type Taq, Klentaq,Omni Taq, or Omni Klentaq.

Reverse Transcriptase Activity.

Reverse transcriptase (RT) activity can create a single-strandedcomplementary DNA (cDNA) from an RNA template.

RT activity can allow reverse transcription of a target RNA into its DNAcomplement. The newly synthesized cDNA can used as a template foramplification using PCR or LAMP, or another DNA amplification protocol.

RT activity can be measured in a variety of protocols known to the art(see e.g., King and O'Connel (2002) RT-PCR Protocols, 1^(st) Ed., HumanPress, ISBN-10 0896038750; Blotter et al. 2013 Angewandte ChemieInternational Edition 52, 11935-11939; Ong et al. 2006 Journal ofMolecular Biology 361, 537-550; Example 1; Example 2).

While other studies have identified different combinations of four aminoacid changes that demonstrate reverse transcriptase activity forKlentaq1 (Blatter et al. 2013 Angewandte Chemie International Edition52, 11935-11939) or Taq DNA polymerase (Ong et al. 2006 Journal ofMolecular Biology 361, 537-550), such mutations are different from anddo not overlap mutations disclosed herein.

Strand Displacement Activity.

A phenotype of a mutant polymerase described herein can have polymeraseactivity (or resistant polymerase activity) and RT activity or stranddisplacement activity.

Strand displacement activity can be measured in a variety of protocolsknown to the art (see e.g., Example 1; Example 2).

Given the well-known lack of strand displacement activity in Taq (SEQ IDNO: 1) or Klentaq1 (SEQ ID NO: 2), strand-displacement and LAMPcatalysis associated with mutation D732N was a very surprisingdiscovery.

Mechanism

While under no obligation to do so, and in no way limiting the scope ofthe present disclosure, the following discussion is directed tomechanism of action. The location of the D732N mutation is neither nearthe active site nor near the DNA in the published crystal structureswith primer and template. It is presently thought that the D732Nmutation may be near the displaced DNA strand duringstrand-displacement, or the incoming RNA template may track indifferently than does DNA template in the crystal structures. Thislocation, or near it, can be a newly reasonable place to put afluorescent probe of template-strand bases.

Although mutation D732N appears distant from the primer and templatestrand in the crystal structures, strand-displacement involves threestrands, and there is yet no information about the track taken by thedisplaced strand. The single-strand template takes a surprising rightturn near 742,743 in the Bst crystal structure 1L3S, so the displacedstrand could also be in this area, since it was just recently unpairedfrom the template strand. That could put it near amino acid residue 732(per wild type Taq numbering). Consistent with this reasoning, Klentaq1(SEQ ID NO: 2) has a positively charged surface RRR (and Bst has KQK) inthe spatially extrapolated position at 715-717 where positively chargedresidues could interact with phosphates of the displaced strand, if itis still close to the enzyme.

PCR

A mutant polymerase (including all variants thereof) described hereincan be used in a variety of polymerase reactions known to the art (seee.g., Dorak (2006) Real-Time PCR, Taylor & Francis, ISBN 041537734X;Bustin, ed. (2004) A-Z of Quantitative PCR, International UniversityLine, ISBN 0963681788; King and O'Connel (2002) RT-PCR Protocols, 1^(st)Ed., Human Press, ISBN-10 0896038750). For example, a mutant polymerasecan be employed in PCR reactions, primer extension reactions, etc.

For example, a mutant polymerase described herein can be used in nucleicacid amplification processes (either alone or in combination with one ormore other enzymes), such as Allele-specific PCR; Assembly PCR orPolymerase Cycling Assembly; Asymmetric PCR;Linear-After-The-Exponential-PCR; Helicase-dependent amplification;Hot-start-PCR; Intersequence-specific PCR; Inverse PCR;Ligation-mediated PCR; Methylation-specific PCR; Miniprimer PCR;Multiplex Ligation-dependent Probe Amplification; Multiplex-PCR; NestedPCR; Overlap-extension PCR; Quantitative PCR; Quantitative End-PointPCR; Quantitative Real-Time PCR; RT-PCR (Reverse Transcription PCR);loop-mediated isothermal amplification (LAMP), reverse transcriptaseloop-mediated isothermal amplification (RT-LAMP), Solid Phase PCR;Thermal asymmetric interlaced PCR; Touchdown PCR; PAN-AC; Universal FastWalking; Long PCR; Rapid Amplified Polymorphic DNA Analysis; RapidAmplification of cDNA Ends (RACE); Differential Display PCR; In situPCR; High-Fidelity PCR; PCR or DNA Sequencing (cycle sequencing).

A target nucleic acid of a sample can be any target nucleic acid ofinterest. For example, a target nucleic acid can be a deoxyribonucleicacid (DNA), a ribonucleic acid (RNA), or an artificial nucleic acidanalog (e.g., a peptide nucleic acid, morpholino- and locked nucleicacid, glycol nucleic acid, or threose nucleic acid).

A primer is understood to refer to an oligonucleotide, whether occurringnaturally or produced synthetically, which is capable of acting as apoint of initiation of nucleic acid synthesis when placed underconditions in which synthesis of a primer extension product which iscomplementary to a nucleic acid strand is induced, e.g., in the presenceof four different nucleotide triphosphates and thermostable enzyme in anappropriate buffer (“buffer” includes pH, ionic strength, cofactors,etc.) and at a suitable temperature. The primer is preferablysingle-stranded for maximum efficiency in amplification, but mayalternatively be double-stranded. If double-stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the thermostableenzyme. The exact lengths of the primers will depend on many factors,including temperature, source of primer and use of the method. Forexample, depending on the complexity of the target sequence, theoligonucleotide primer typically contains 15-25 nucleotides, although itmay contain more or few nucleotides. Short primer molecules generallyrequire colder temperatures to form sufficiently stable hybrid complexeswith template.

A target nucleic acid, e.g., a template DNA molecule, is understood tobe a strand of a nucleic acid from which a complementary nucleic acidstrand can be synthesized by a DNA polymerase, for example, in a primerextension reaction.

A mutant polymerase described herein can be used in an end-point PCR.For example, end-point PCR is commonly carried out in a reaction volumeof about 10-200 μl in small reaction tubes (about 0.2-0.5 ml volumes) ina thermal cycler. A mutant polymerase described herein can be used witha variety of commercially available end-point PCR kits. The use of amutant polymerase enzyme described herein generally does not requireany, or substantial, changes in the typical end-point PCR protocol, butcan allow, for example, a sample having a higher amount of an inhibitorysubstance.

A mutant polymerase described herein can be used in real-time PCR (alsoknown as a quantitative polymerase chain reaction (qPCR)). For example,a mutant polymerase described herein can be used in a real-time PCRassay featuring a non-specific fluorescent dye (e.g., a fluorochrome)that can intercalate with any double-stranded DNA. With a non-specificfluorescent dye, an increase in DNA product during PCR can lead to anincrease in fluorescence intensity and is measured at each cycle, thusallowing DNA concentrations to be quantified.

As another example, a mutant polymerase described herein can be used ina real-time PCR assay featuring a hybridization probe. As anotherexample, a mutant polymerase described herein can be used in a real-timePCR multiplex assay featuring a hybridization probe. A hybridizationprobe can be a sequence-specific DNA probe including a fluorescentreporter at one end and a quencher of fluorescence at the opposite endof the probe, where break down of the probe by a 5′ to 3′ exonucleaseactivity of a polymerase can break the reporter-quencher proximity andthus allow unquenched emission of fluorescence, which can be detectedafter excitation with a laser (e.g., a TaqMan® assay). With ahybridization probe, an increase in the product targeted by the reporterprobe at each PCR cycle can cause a proportional increase influorescence due to the breakdown of the probe and release of thereporter. A mutant polymerase described herein can be used with avariety of commercially available real-time PCR kits.

RT-PCR.

As another example, a mutant polymerase described herein can be usedReverse Transcriptase (RT) PCR. In some embodiments, the use of a mutantpolymerase enzyme described herein does not require any, or substantial,changes in the typical protocol, but can allow, for example, exclusionof a reverse transcriptase enzyme from a Reverse-Transcriptase (RT) PCR.

In some embodiments, the use of a mutant polymerase enzyme describedherein does not require any, or substantial, changes in the typicalprotocol, but can allow, for example, exclusion of a reversetranscriptase enzyme from a Reverse-Transcriptase (RT) PCR in an amountsufficient to generate cDNA from an RNA template or in an amountsufficient to initiate reverse transcription.

A mutant polymerase described herein can be used in a variety of RT-PCRprotocols known to the art (see e.g., King and O'Connel (2002) RT-PCRProtocols, 1^(st) Ed., Human Press, ISBN-10 0896038750). The use of themutant polymerase enzymes described herein generally does not requireany, or substantial, changes in the typical protocol, other than, forexample, being able to eliminate a separate reverse transcriptase enzymefrom the reaction mixture.

Conventionally, RT-PCR can be achieved as either a one-step or atwo-step reaction. In the one-step approach, the entire reaction fromcDNA synthesis to PCR amplification can occur in a single tube. Theconventional two-step reaction can require that the reversetranscriptase reaction and PCR amplification be performed in separatetubes. Mutant polymerases described herein can be used in a one-stepreaction or a two-step reaction. For example, a mutant polymerasedescribed herein can be used in a one-step RT-PCR. As another example, amutant polymerases described herein can be used in a two-step RT-PCR.

An exemplary RT-PCR is described below. In some embodiments, a one-stepRT-PCR can take mRNA targets (e.g., up to 6 kb) and subject them toreverse transcription and then PCR amplification in a single test tube.A sequence-specific primer can be selected. A reaction mixture isprepared, including, e.g., dNTPs, primers, template RNA, necessaryenzymes and a buffer solution. According to the present disclosure, amutant polymerase described herein can have both DNA polymerase activityand reverse transcriptase activity, which can eliminate the need for twoseparate enzymes. The reaction mixture can be added to a PCR tube foreach reaction, to which can be added template RNA. PCR tubes can beplaced in a thermal cycler to begin cycling. The first cycle can bereverse transcription to synthesize cDNA. The (optional) second cyclecan be initial denaturation. Conventionally, the denaturation step caninactivate a reverse transcriptase enzyme but a mutant polymerasedisclosed herein can be resistant to such denaturation and so, the stepmay not be necessary. The next cycles (e.g., 40 to 50) can be anamplification program, consisting of three steps: denaturation,annealing, elongation. The RT-PCR products can then be analyzedaccording to conventional techniques (e.g., gel electrophoresis).

An RT-PCR can be an end-point RT-PCR or a real-time RT-PCR. ConventionalRT-PCR protocols, such as end-point RT-PCR or real-time RT-PCR, can beused in combination with, or adapted to, mutant polymerases and methodsdescribed herein.

An end-point RT-PCR can require the detection of target nucleic acidlevels by the use of, e.g., fluorescent dyes like ethidium bromide, P32labeling of PCR products using phosphorimager, or by scintillationcounting. End-point RT-PCR can be achieved using three differentmethods: relative, competitive and comparative. Relative quantificationsof RT-PCR can involve the co-amplification of an internal controlsimultaneously with the gene of interest. The internal control can beused to normalize the samples. Competitive RT-PCR technique can be usedfor absolute quantification, and can use a synthetic “competitor” RNAthat can be distinguished from the target RNA by a small difference insize or sequence. Comparative RT-PCR is similar to competitive RT-PCR inthat the target RNA competes for amplification reagents within a singlereaction with an internal standard of unrelated sequence.

Real-time RT-PCR can use fluorescent DNA probes (e.g., SYBR Green,TaqMan, Molecular Beacons, or Scorpions).

It is noted that reverse transcriptase (RT) PCR is not to be confusedwith real-time polymerase chain reaction (Q-PCR), which is sometimes(incorrectly) abbreviated as RT-PCR in the art. In RT-PCR, an RNA strandis first reverse transcribed into its DNA complement (complementary DNA,or cDNA) using the enzyme reverse transcriptase, and the resulting cDNAis amplified using traditional PCR. Like with end-point PCR,conventional RT-PCR protocols require extensive purification steps priorto amplification to purify RNA from inhibitors and ribonucleases, whichcan destroy the RNA template. Both the inhibition and degradation of RNAare major concerns in important clinical and diagnostics tests, whichmay lead to false-negative results.

Applications of RT-PCR include, but are not limited to, detection of RNAvirus pathogens; analysis of mRNA expression patterns of certain genesrelated to various diseases; semiquantitative determination of abundanceof specific different RNA molecules within a cell or tissue as a measureof gene expression; and cloning of eukaryotic genes in prokaryotes.

U.S. Patent Application Publication No. 2014/0113299 describes a mutantpolymerase having a D732N mutation “used in combination with an enzymehaving reverse transcriptase activity in a real-time reversetranscriptase (RT) PCR amplification of an RNA target.” But U.S. PatentApplication Publication No. 2014/0113299 does not describe an RT-PCRreaction that includes a mutant polymerase described herein but does notrequire or does not include a separate enzyme having reversetranscriptase activity.

LAMP.

As another example, a mutant polymerase described herein can be used tocatalyze loop-mediated isothermal amplification (LAMP) of a DNA target.In contrast to PCR in which the reaction is carried out with a series ofalternating temperature steps or cycles, isothermal amplification iscarried out at a constant temperature, and does not require a thermalcycler. Bst large fragment DNA polymerase is often used for LAMP butdenatures at 65-70° C. and therefore cannot catalyze PCR. Althoughfull-length, wild-type Taq (SEQ ID NO: 1) can strand-displace to someextent, its 5′-endonuclease (flap endonuclease, FEN) cleaves thedisplaced strand so much that it is not appropriate for LAMP.

In LAMP, a target sequence can be amplified at a constant temperature ofabout 60-using either two or three sets of primers and a polymerase withhigh strand displacement activity in addition to a replication (i.e.,polymerase) activity (e.g., Bst large fragment DNA polymerase). Asdescribed herein, a mutant polypeptide having DNA polymerase activityand strand displacement activity can be used for LAMP, in place of or inaddition to the relatively sensitive Bst polymerase.

Conventionally, four different primers can used to identify six distinctregions on a target gene, which can increase specificity. An additionalpair of “loop primers” can further accelerate the conventional LAMPreaction. Due to the specific nature of the action of these primers, theamount of DNA produced in LAMP can be considerably higher than PCR basedamplification. LAMP primer design can be according to well knownprocesses in the art. Except as otherwise noted herein, therefore, theprocess of the present disclosure can be carried out in accordance withknown LAMP protocols.

The product of LAMP can be a series of concatemers of the target region,giving rise to a characteristic “ladder” or banding pattern on a gel,rather than a single band as with PCR. Detection of a LAMP amplificationproduct can be determined according to well known processes in the art.Except as otherwise noted herein, therefore, the process of the presentdisclosure can be carried out in accordance with such processes. Forexample, detection of a LAMP amplification product can be determinedusing photometry for turbidity caused by an increasing quantity ofmagnesium pyrophosphate precipitate in solution as a byproduct ofamplification. This can allow visualization by the naked eye, especiallyfor larger reaction volumes, or via simple detection approaches forsmaller volumes. As another example, the reaction can be followed inreal-time by measuring the turbidity or by fluorescence using anintercalating dyes (e.g., SYTO 9, SYBR green, Eva Green) can be used tocreate a visible color change, which can be visually detected ormeasured by instrumentation. LAMP can be quantitative because dyemolecules intercalate or directly label the DNA, and in turn can becorrelated to the number of copies initially present. As anotherexample, in-tube detection of DNA amplification can use manganese loadedcalcein which starts fluorescing upon complexation of manganese bypyrophosphate during in vitro DNA synthesis.

RT-LAMP.

As another example, a mutant polymerase described herein can be used tocatalyze loop-mediated isothermal amplification using RNA template(RT-LAMP). Conventional RT-LAMP requires primers, a reversetranscriptase enzyme, and a DNA polymerase enzyme having stranddisplacement activity for the amplification of RNA. Similar to RT-PCR,conventional RT-LAMP requires a reverse transcriptase enzyme (notnecessary according to methods and mutant polymerases described herein)to synthesize complementary DNA (cDNA) from RNA sequences. This cDNA canthen be amplified using DNA polymerase. As described herein, use of amutant polymerase can eliminate the need for a separate reversetranscriptase enzyme is not required.

RT-LAMP can be desirable because of the relatively low reactiontemperature and no need for thermocycling equipment necessary for othermethods like PCR.

In conventional LAMP, four specially designed primers can recognizedistinct target sequences on a template strand. Such primers bind onlyto these sequences which allows for high specificity. Out of the fourprimers involved, two of them are “inner primers” (FIP and BIP),designed to synthesize new DNA strands. The outer primers (F3 and B3)anneal to the template strand and also generate new DNA. These primersare accompanied by a DNA polymerase which can aid strand displacementand can release the newly formed DNA strands.

The BIP primer (in conventional methods, accompanied by a reversetranscriptase enzyme, which is not required with mutant polymerasesdescribed herein), can initiate the process by binding to a targetsequence on the 3′ end of an RNA template and synthesizing a copy DNAstrand. The B3 primer can also bind the 3′ end and along with apolypeptide having DNA polymerase activity (e.g., a mutant polymerasedescribed herein) can simultaneously create a new cDNA strand whiledisplacing the previously made copy. The double stranded DNA containingthe template strand is no longer needed.

At this point, the single stranded copy can loop at the 3′ end as itbinds to itself. The FIP primer can bind to the 5′ end of this singlestrand and accompanied by a polypeptide having DNA polymerase activity(e.g. a mutant polymerase described herein), can synthesize acomplementary strand. The F3 primer, with DNA polymerase, can bind tothis end and can generate a new double stranded DNA molecule whiledisplacing the previously made single strand.

This newly displaced single strand can act as the starting point for aLAMP cycling amplification. The DNA can have a dumbbell-like structureas the ends fold in and self anneal. This structure can become astem-loop when the FIP or BIP primer once again initiates DNA synthesisat one of the target sequence locations. This cycle can be started fromeither the forward or backward side of the strand using an appropriateprimer. Once this cycle has begun, the strand can undergo self-primedDNA synthesis during the elongation stage of the amplification process.As described above, this amplification can take place in about an hour,under isothermal conditions between about 60-65° C.

A mutant polymerase described herein can be used in RT-LAMP and notrequire the presence of a separate reverse transcriptase enzyme. In someembodiments, a mutant polymerase described herein is used in RT-LAMP anda separate reverse transcriptase enzyme is not present in the reactionmixture.

It has also been discovered that a Bst large fragment DNA polymerasecomprises reverse transcriptase activity. In some embodiments, RT-LAMPcan be performed with a Bst large fragment DNA polymerase (e.g., Mantaor Bst 2.0 from Enzymatics, Bst 1.0 from New England Biolabs). In someembodiments, a Bst large fragment DNA polymerase (e.g., Manta or Bst 2.0from Enzymatics, Bst 1.0 from New England Biolabs) is used in RT-LAMPand a separate reverse transcriptase enzyme is not present in thereaction mixture. In some embodiments, a Bst large fragment DNApolymerase comprising a sequence of SEQ ID NO: 10, or a variant at least95% identical thereto having DNA polymerase activity, reversetranscriptase activity, and strand displacement activity, is used inRT-LAMP and a separate reverse transcriptase enzyme is not present inthe reaction mixture. In some embodiments, a RT-LAMP reaction mixtureincludes both a mutant polymerase disclosed herein and a Bst largefragment DNA polymerase (e.g., (Manta or Bst 2.0 from Enzymatics, Bst1.0 from New England Biolabs) (e.g., a Bst large fragment DNA polymerasehaving a sequence of SEQ ID NO: 10, or a variant at least 95% identicalthereto having DNA polymerase activity, reverse transcriptase activity,and strand displacement activity).

Thus, methods and compositions described herein can be applied toimprove the nucleic acid detection in RT-PCR, LAMP, or RT-LAMP. BothRT-PCR and RT-LAMP can benefit from high temperature tissue and virusdisruption, and higher temperature reaction, to improve the convenienceand selectivity of RNA detection, such as for Ebola and Dengue. A mutantpolymerase described herein can allow such higher temperatures andremove the requirement for a separate RT enzyme in the reaction.

The buffer for use in the various PCR assay mixtures described herein isgenerally a physiologically compatible buffer that is compatible withthe function of enzyme activities and enables cells or biologicalmacromolecules to retain their normal physiological and biochemicalfunctions. Typically, a physiologically compatible buffer will include abuffering agent (e.g., TRIS, MES, PO₄, HEPES, etc.), a chelating agent(e.g., EDTA, EGTA, or the like), a salt (e.g., ammonium sulfate, NaCl,KCl, MgCl₂, CaCl₂, NaOAc, KOAc, Mg(OAc)₂, etc.) and optionally astabilizing agent (e.g., sucrose, glycerine, Tween20, etc.).

Various PCR additives and enhancers can be employed with the methodsdescribed herein. For example, betaine (e.g., MasterAmp™ 10×PCR,Epicentre Biotechnologies) can be added to the PCR assay. Betaine can beincluded at final concentration about 0.75 M to about 2 M.

As another example, a mutant polymerase described herein can be used inconjunction with a PCR enhancer described in US Pat Pub No. 2012/0028259or WO 2012/088479, each incorporated herein by reference. For example, amutant polymerase can be used in conjunction with a PCR enhancerincluding trehalose (e.g., about 0.1 to about 1.0 M D-(+)-trehalose peramplification reaction mixture volume), carnitine (about 0.1 to about1.5 M L-carnitine per amplification reaction mixture volume), or anon-ionic detergent (e.g., NP-40, IGEPAL® CA-630, BRIJ™-58, TWEEN™-20,or TRITON™ X-100 at about 0.01% to about 8% non-ionic detergent peramplification reaction mixture volume) or optionally one or more ofheparin (e.g., an amount of heparin equivalent to about 2 units to about50 units heparin per mL of whole blood, plasma, or serum in anamplification reaction mixture), casein (at least about 0.05% up toabout 2.5% per amplification reaction mixture volume), orpolyvinylpyrrolidone (PVP) or a modified polymer of PVP (PVPP) (e.g.,about 0.1% up to about 25%). As another example, a mutant polymerase canbe used in conjunction with a PCR enhancer including about 0.6 Mtrehalose per amplification reaction mixture volume; about 0.5 Mcarnitine per amplification reaction mixture volume; or a non-ionicdetergent (e.g., a polyoxyethylene cetyl ether at about 0.04% to about0.2% or a nonyl phenoxylpolyethoxylethanol at about 0.4% to about 0.8%per amplification reaction mixture volume); or optional heparin at about10 units per mL of whole blood, blood fraction, plasma, or serum.

As another example, a mutant polymerase described herein can be used inconjunction with commercially available PCR amplification reactionenhancers, such as MasterAmp™ 10×PCR Enhancer, EpicentreBiotechnologies; TaqMaster PCR Enhancer, MasterTaq Kit, PCR ExtenderSystem, 5 PRIME GmbH; Hi-Spec Additive, Bioline; PCRboost™, Biomatrica®;PCRX Enhancer System, Invitrogen; Taq Extender™ PCR Additive, PerfectMatch® PCR Enhancer, Stratagene; Polymer-Aide PCR Enhancer,Sigma-Aldrich.

Chemistry and Molecular Engineering

The following definitions and methods are provided to better define thepresent disclosure and to guide those of ordinary skill in the art inthe practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

A chemical bond is understood as an attraction between atoms of abiomolecule and atoms of a matrix material that allows the formation ofa linkage between atoms (e.g., atoms of the same molecule or differentmolecules). A bond can be caused by an electrostatic force of attractionbetween opposite charges, either between electrons and nuclei, or as theresult of a dipole attraction. A bond can be, for example, a covalentbond, a coordinate covalent bond, an ionic bond, polar covalent, adipole-dipole interaction, a London dispersion force, a cation-piinteraction, or hydrogen bonding.

Compositions and methods described herein utilizing molecular biologyprotocols can be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russell (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005)Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production ofRecombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein ExpressionTechnologies, Taylor & Francis, ISBN-10: 0954523253).

A mutation refers to a change introduced into a parental sequence,including, but not limited to, substitutions, insertions, or deletions(including truncations). The consequences of a mutation include, but arenot limited to, the creation of a new character, property, function,phenotype or trait not found in the protein encoded by the parentalsequence.

Enzyme activity refers to the specificity and efficiency of a DNApolymerase. Enzyme activity of a DNA polymerase can also be referred toas polymerase activity, which typically refers to the activity of a DNApolymerase in catalyzing the template-directed synthesis of apolynucleotide. Enzyme activity of a polymerase can be measured usingvarious techniques and methods known in the art. For example, serialdilutions of polymerase can be prepared in dilution buffer. The reactionmixtures can be incubated at, e.g., 74° C. and stopped by cooling to,e.g., 40° C. and adding ice-cold EDTA. An aliquot can be removed fromeach reaction mixture. Unincorporated radioactively labeled dCTP can beremoved from each aliquot by gel filtration (e.g., Centri-Sep, PrincetonSeparations, Adelphia, N. J.). The column eluate can be mixed withscintillation fluid. Radioactivity in the column eluate can bequantified with a scintillation counter to determine the amount ofproduct synthesized by the polymerase. One unit of polymerase activitycan be defined as the amount of polymerase necessary to synthesize 10nmole of product in 30 minutes (see e.g., Lawyer et al. 1989 J. Biol.Chem. 264, 6427-647). Other methods of measuring polymerase activity areknown in the art (see e.g. Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual, 3d ed., Cold Spring Harbor LaboratoryPress, ISBN-10: 0879695773).

The terms “heterologous DNA sequence”, “exogenous DNA segment” or“heterologous nucleic acid,” as used herein, each refer to a sequencethat originates from a source foreign to the particular host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides. A “homologous”DNA sequence is a DNA sequence that is naturally associated with a hostcell into which it is introduced.

Expression vector, expression construct, plasmid, or recombinant DNAconstruct is generally understood to refer to a nucleic acid that hasbeen generated via human intervention, including by recombinant means ordirect chemical synthesis, with a series of specified nucleic acidelements that permit transcription or translation of a particularnucleic acid in, for example, a host cell. The expression vector can bepart of a plasmid, virus, or nucleic acid fragment. Typically, theexpression vector can include a nucleic acid to be transcribed operablylinked to a promoter.

A “promoter” is generally understood as a nucleic acid control sequencethat directs transcription of a nucleic acid. An inducible promoter isgenerally understood as a promoter that mediates transcription of anoperably linked gene in response to a particular stimulus. A promotercan include necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter can optionally include distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

A “transcribable nucleic acid molecule” as used herein refers to anynucleic acid molecule capable of being transcribed into a RNA molecule.Methods are known for introducing constructs into a cell in such amanner that the transcribable nucleic acid molecule is transcribed intoa functional mRNA molecule that is translated and therefore expressed asa protein product. Constructs may also be constructed to be capable ofexpressing antisense RNA molecules, in order to inhibit translation of aspecific RNA molecule of interest. For the practice of the presentdisclosure, conventional compositions and methods for preparing andusing constructs and host cells are well known to one skilled in the art(see e.g., Sambrook and Russel (2006) Condensed Protocols from MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in MolecularBiology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook andRussel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., ColdSpring Harbor Laboratory Press, ISBN-10: 0879695773; Green and Sambrook2012 Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring HarborLaboratory Press, ISBN-10: 1605500569; Elhai, J. and Wolk, C. P. 1988.Methods in Enzymology 167, 747-754).

The “transcription start site” or “initiation site” is the positionsurrounding the first nucleotide that is part of the transcribedsequence, which is also defined as position +1. With respect to thissite all other sequences of the gene and its controlling regions can benumbered. Downstream sequences (i.e., further protein encoding sequencesin the 3′ direction) can be denominated positive, while upstreamsequences (mostly of the controlling regions in the 5′ direction) aredenominated negative.

“Operably-linked” or “functionally linked” refers preferably to theassociation of nucleic acid sequences on a single nucleic acid fragmentso that the function of one is affected by the other. For example, aregulatory DNA sequence is said to be “operably linked to” or“associated with” a DNA sequence that codes for an RNA or a polypeptideif the two sequences are situated such that the regulatory DNA sequenceaffects expression of the coding DNA sequence (i.e., that the codingsequence or functional RNA is under the transcriptional control of thepromoter). Coding sequences can be operably-linked to regulatorysequences in sense or antisense orientation. The two nucleic acidmolecules may be part of a single contiguous nucleic acid molecule andmay be adjacent. For example, a promoter is operably linked to a gene ofinterest if the promoter regulates or mediates transcription of the geneof interest in a cell.

A “construct” is generally understood as any recombinant nucleic acidmolecule such as a plasmid, cosmid, virus, autonomously replicatingnucleic acid molecule, phage, or linear or circular single-stranded ordouble-stranded DNA or RNA nucleic acid molecule, derived from anysource, capable of genomic integration or autonomous replication,comprising a nucleic acid molecule where one or more nucleic acidmolecule has been operably linked.

A constructs of the present disclosure can contain a promoter operablylinked to a transcribable nucleic acid molecule operably linked to a 3′transcription termination nucleic acid molecule. In addition, constructscan include but are not limited to additional regulatory nucleic acidmolecules from, e.g., the 3′-untranslated region (3′ UTR). Constructscan include but are not limited to the 5′ untranslated regions (5′ UTR)of an mRNA nucleic acid molecule which can play an important role intranslation initiation and can also be a genetic component in anexpression construct. These additional upstream and downstreamregulatory nucleic acid molecules may be derived from a source that isnative or heterologous with respect to the other elements present on thepromoter construct.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.

“Transformed,” “transgenic,” and “recombinant” refer to a host cell ororganism such as a bacterium, cyanobacterium, animal or a plant intowhich a heterologous nucleic acid molecule has been introduced. Thenucleic acid molecule can be stably integrated into the genome asgenerally known in the art and disclosed (Sambrook 1989; Innis 1995;Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, butare not limited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like. Theterm “untransformed” refers to normal cells that have not been throughthe transformation process.

“Wild-type” refers to a virus or organism found in nature without anyknown mutation.

Design, generation, and testing of the variant nucleotides, and theirencoded polypeptides, having the above required percent identities andretaining a required activity of the expressed protein is within theskill of the art. For example, directed evolution and rapid isolation ofmutants can be according to methods described in references including,but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688;Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) ProcNatl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art couldgenerate a large number of nucleotide or polypeptide variants having,for example, at least 95-99% identity to the reference sequencedescribed herein and screen such for desired phenotypes according tomethods routine in the art.

Nucleotide or amino acid sequence identity percent (%) is understood asthe percentage of nucleotide or amino acid residues that are identicalwith nucleotide or amino acid residues in a candidate sequence incomparison to a reference sequence when the two sequences are aligned.To determine percent identity, sequences are aligned and if necessary,gaps are introduced to achieve the maximum percent sequence identity.Sequence alignment procedures to determine percent identity are wellknown to those of skill in the art. Often publicly available computersoftware such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software isused to align sequences. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared. When sequences are aligned, the percentsequence identity of a given sequence A to, with, or against a givensequence B (which can alternatively be phrased as a given sequence Athat has or comprises a certain percent sequence identity to, with, oragainst a given sequence B) can be calculated as: percent sequenceidentity=X/Y100, where X is the number of residues scored as identicalmatches by the sequence alignment program's or algorithm's alignment ofA and B and Y is the total number of residues in B. If the length ofsequence A is not equal to the length of sequence B, the percentsequence identity of A to B will not equal the percent sequence identityof B to A.

Generally, conservative substitutions can be made at any position solong as the required activity is retained. So-called conservativeexchanges can be carried out in which the amino acid which is replacedhas a similar property as the original amino acid, for example theexchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser byThr. Deletion is the replacement of an amino acid by a direct bond.Positions for deletions include the termini of a polypeptide andlinkages between individual protein domains. Insertions areintroductions of amino acids into the polypeptide chain, a direct bondformally being replaced by one or more amino acids. Amino acid sequencecan be modulated with the help of art-known computer simulation programsthat can produce a polypeptide with, for example, improved activity oraltered regulation. On the basis of this artificially generatedpolypeptide sequences, a corresponding nucleic acid molecule coding forsuch a modulated polypeptide can be synthesized in-vitro using thespecific codon-usage of the desired host cell.

“Highly stringent hybridization conditions” are defined as hybridizationat 65° C. in a 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 Msodium citrate). Given these conditions, a determination can be made asto whether a given set of sequences will hybridize by calculating themelting temperature (T_(m)) of a DNA duplex between the two sequences.If a particular duplex has a melting temperature lower than 65° C. inthe salt conditions of a 6×SSC, then the two sequences will nothybridize. On the other hand, if the melting temperature is above 65° C.in the same salt conditions, then the sequences will hybridize. Ingeneral, the melting temperature for any hybridized DNA:DNA sequence canbe determined using the following formula: T_(m)=81.5° C.+16.6(log₁₀[Na⁺])+0.41(fraction G/C content)−0.63(% formamide)−(600/I).Furthermore, the T_(m) of a DNA:DNA hybrid is decreased by 1-1.5° C. forevery 1% decrease in nucleotide identity (see e.g., Sambrook andRussell, 2006).

Host cells can be transformed using a variety of standard techniquesknown to the art (see, e.g., Sambrook and Russell (2006) CondensedProtocols from Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002)Short Protocols in Molecular Biology, 5th ed., Current Protocols,ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10:0879695773; Green and Sambrook 2012 Molecular Cloning: A LaboratoryManual, 4th ed., Cold Spring Harbor Laboratory Press, ISBN-10:1605500569; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167,747-754). Such techniques include, but are not limited to, viralinfection, calcium phosphate transfection, liposome-mediatedtransfection, microprojectile-mediated delivery, receptor-mediateduptake, cell fusion, electroporation, and the like. The transfectedcells can be selected and propagated to provide recombinant host cellsthat comprise the expression vector stably integrated in the host cellgenome.

Exemplary nucleic acids which may be introduced to a host cell include,for example, DNA sequences or genes from another species, or even genesor sequences which originate with or are present in the same species,but are incorporated into recipient cells by genetic engineeringmethods. The term “exogenous” is also intended to refer to genes thatare not normally present in the cell being transformed, or perhapssimply not present in the form, structure, etc., as found in thetransforming DNA segment or gene, or genes which are normally presentand that one desires to express in a manner that differs from thenatural expression pattern, e.g., to over-express. Thus, the term“exogenous” gene or DNA is intended to refer to any gene or DNA segmentthat is introduced into a recipient cell, regardless of whether asimilar gene may already be present in such a cell. The type of DNAincluded in the exogenous DNA can include DNA which is already presentin the cell, DNA from another individual of the same type of organism,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

Host strains developed according to the approaches described herein canbe evaluated by a number of means known in the art (see e.g., Studier(2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005)Production of Recombinant Proteins: Novel Microbial and EukaryoticExpression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004)Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Kits

Also provided are kits. Such kits can include an agent or compositiondescribed herein and, in certain embodiments, instructions foradministration. Such kits can facilitate performance of the methodsdescribed herein. When supplied as a kit, the different components ofthe composition can be packaged in separate containers and admixedimmediately before use. Components include, but are not limited to amutant polymerase described herein or a nucleic acid encoding suchmutant polymerase or, optionally, a primer, a buffer, or othercomposition or component (e.g., a magnesium salt) necessary or helpfulfor PCR. Such packaging of the components separately can, if desired, bepresented in a pack or dispenser device which may contain one or moreassay unit forms containing a composition. The pack may, for example,comprise metal or plastic foil such as a blister pack. Such packaging ofthe components separately can also, in certain instances, permitlong-term storage without losing activity of the components.

Kits may also include reagents in separate containers such as, forexample, sterile water or saline to be added to a lyophilized activecomponent packaged separately. For example, sealed glass ampules maycontain a lyophilized component and in a separate ampule, sterile water,sterile saline or sterile each of which has been packaged under aneutral non-reacting gas, such as nitrogen. Ampules may consist of anysuitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include bottles that may be fabricated from similarsubstances as ampules, and envelopes that may consist of foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, and the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to mix. Removable membranes may beglass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate, ormay be supplied as an electronic-readable medium, such as a floppy disc,mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and thelike. Detailed instructions may not be physically associated with thekit; instead, a user may be directed to an Internet web site specifiedby the manufacturer or distributor of the kit.

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1

This study showed the isolation of mutants resistant to inhibitors offood assays thus eliminating or decreasing the time and extent of theseextraction procedures or improve the reliability of PCR assays of food.Total E. coli cells were used as a source of enzyme to screen circa4,000 mutagenized clones that expressed Taq DNA polymerase from aplasmid. The screen used crude extract as the enzyme and template.Expression of 50 μl cultures in 96-well plates was induced, and PCRprimers for endogenous ribosomal DNA (385 bp product) (forwardTACAGACGTTTAAGCTTCGCAATTACC GGTT (SEQ ID NO: 11) and reverseAAAAAGCTGCAAATTGCGGTAGGTATTATT (SEQ ID NO: 12)), reaction buffer andSYBR Green were added as 35 μl directly to 3-5 μl of the bacterialculture, followed by immediate temperature cycling. Wild-type Taq orKlentaq1 clones give rise to robust PCR amplification under theseconditions. When chocolate or cracked-pepper extract (3 μl of 10% w/v)was also included, PCR was suppressed for all but a few clones. At afrequency of about 1% in this mutagenized library, real-time PCRanalysis identified enzyme variants that could still catalyze PCR in thepresence of inhibitors. The cracked pepper screen gave rise to cloneA111 (FIG. 20 ) and subsequent DNA sequencing showed only a singlechange, D732N. Resistance to chocolate, blood, and bile was subsequentlyobserved for enzyme A111.

Cycling conditions: Pre-heat: both 10′/95° C. and 5′/95° C. work,followed by 30 sec/95° C., 40 sec/54° C. and 3 min/70° C., for 40-42cycles. Melting curve analysis in the interval 60-90° C. followed theamplification.

Cycler used: Opticon-2, BioRad. Reactions contained 0.6×SYBR Green, and2-3 μl 10% black pepper extract per 35 μl reaction volume.

The target for these inhibition tests is in the human cross-link repair1A gene. Human DNA was supplied at 10 ng per 50 μl reaction volume inthe no-inhibitor controls and the food reactions. No exogenous templateDNA was added to the blood reactions. The amplicon size is 976 bp, andthe primers sequences are For: ctaccaaggtaatgaagcaaatggatat (SEQ ID NO:13); Rev: tggcagcaaccaaagtatatgaaaggg (SEQ ID NO: 14). Taq 732N enzymestock concentration was 0.8 OD280, and the amount used varied asdescribed. Taq WT was supplied by NE Biolabs and used at 0.8 μl perreaction. PCR reaction buffer used at 1× was diluted from 10× stock 500mM Tris-CI, pH 9.1, 160 mM ammonium sulfate, 0.25% Brij 58, and 25 mMmagnesium chloride. The PCR cycling program was 94° C. 10 minutes once;35 cycles of 94° C. 40 second, 60° C. 1 minute, 70° C. 3 min.

Bar chocolate (Lindt, 70% cocoa) was suspended at 10% w/v in water usinga bead-beater and clarified by centrifugation. The supernate was thendiluted with reaction mix to final amounts per 50 μl reaction of 0,0.15, 0.3, 0.6, and 1.2 μl, increasing left to right, as indicated. TaqD732N enzyme used was only 0.04 μl for the zero inhibitor control, sinceit was found to cause smearing at this pH if more was used. Wheninhibitor was present, 0.8 μl of Taq D732N was used. Wild-type Taq (NEBiolabs) was included at 0.8 μl per each reaction. FIG. 17 shows thatTaq D732N produced results at all but the highest amount of chocolate.

Black pepper (Shoppers Value, supermarket) was suspended at 10% w/v inwater and was filtered to remove solids. Tested volumes added to the PCRreactions were 0, 0.063, 0.125, 0.25, 0.5 μl. Whole blood (single-donorValley Biomedical, Virginia, USA.) was stored frozen. Thawed amountsadded to the PCR reactions were 0, 5%, 10%, 15%, 20% of the reactionvolume. FIG. 18 shows that Taq D732N produced results with the first twoconcentrations of black pepper, and all tested concentrations of wholeblood.

Example 2

Half-Dumbbell Construction.

A DNA “handle” was amplified from phage lambda DNA by PCR using primers13.16 (GGCATTGTTTGGTAGGTGAGAGATCT (SEQ ID NO: 15)) and 13.17′(ACAAATGACAAGAGTCTGGTTCAGAAGATA (SEQ ID NO: 16)), precipitated withPEG+SDS, and digested with HindIII and BgIII at its opposite ends. Astem loop consisting of self-annealed, 5′-phosphorylated DNA 11.164(5P-agctCTGTCTCTTATACACATCTaataGTTTAACTTTAAGAAGGAGATATAaataAGAT GTGTATAAGAGACAG (SEQ ID NO: 17)) was formed using T3 ligase (Enzymatics)in T4 ligase buffer (NEB) supplemented with 11 mM NaCl. This product wasprecipitated with PEG+SDS and resuspended in 1× Cutsmart buffer (NEB).To each 1.5 μg of DNA was added 1 μl T7 exonuclease (NEB) on andincubated on ice for 40 minutes to allow removal of ca. 50 bases fromthe 5′-BgIII end, followed by heat denaturation of the exonuclease at75° C. for 15 min. In a buffer of TEN+200 mM NaCl, primer 13.16 wasannealed to replace some of the bases removed by the T7 exonuclease,with a gap in “front” of it at its 3′-end. Klentaq1 is not able tosynthesize any noticeable bases at this gap, but Bst DNA polymeraselarge fragment (Manta from Enzymatics) can extend this 1.2 kb DNAproduct to 2.4 kb by strand-displacement, and then copying the displacedstrand. Compared to the parent (full-length) Taq or Klentaq1 enzymes,mutants D732N of each show a significant ability to produce the 2.4 kbproduct (FIG. 6B).

RT-LAMP MS2 RNA Primer Mixture.

RT-LAMP MS2 RNA primer mixture (TGTCATGGGATCCGGATGTT (SEQ ID NO: 18),CAATAGAGCCGCTCTCAGAG (SEQ ID NO: 19), CCAGAGAGGAGGTTG CCAA (SEQ ID NO:20), TGCAGGATGCAGCGCCTTA (SEQ ID NO: 21), GCCCAAACAACGACGATCGGTAgagtcAAACCAGCATCCGTAGCCT (SEQ ID NO: 22), andGCACGTTCTCCAACGGTGCTgagtcGGTTGCTTGTTCAGCGAACT (SEQ ID NO: 23)) contained120 μl of 1.05× complete reaction buffer (see below), 20 μl of F3 and B3from 10 μM in each stock, and 20 μl of Fip, Bip, FL and BL from 40 μM ineach stock (i.e., ratios 1:4:4), and 1 μl of Antarctic UDG (NE biolabs).Four μl was added to 21-22 μl of otherwise complete reaction volume as ahanging drop hot start (see below).

RT-Lamp Reactions.

RT-LAMP reactions used 7 times less dNTP, and half as much primer,compared to many published LAMP reactions (see e.g., Tomita et al. 2008Nature Protocols 3, 877-882; Tanner et al. 2012 Biotechniques 53, 81-89;Chander et al. 2014 Frontiers in Microbiology, 5). KLA buffer (1×=50 mMTris-HCl pH 8.55, 2.9 mM MgCl₂, 0.025% Brij 58, 8 mM ammonium sulfate)was supplemented with final 0.75 M betaine, 0 or 25 mM KCl, 4 mM DTT,0.1 mM CaCl₂ and 200 μM each of 5 dNTP (A,G,C,T,U). The DTT was includedto allow for efficient thermal inactivation of E. coli RNase I. Thefifth triphosphate, dUTP, was included to allow the potential use ofuracil glycosylase (UDG) to allow removal of contaminating product fromprevious amplifications, but no UDG was used here except in the primermix (see above). Instead calcium-requiring DNase I (NE Biolabs) was usedas follows: At time of LAMP reaction setup, the pre-reaction mix at1.05× and lacking enzyme and nucleic acid, was supplemented with 1/100volume of pancreatic DNAse I stock (NE Biolabs; final 10 μg/ml DNase,0.1 mM CaCl₂) to combat carry-over DNA contamination. The subject DNApolymerase enzyme (about 0.5 to 1 μg or as indicated) to each 21 μl andincubated at room temperature for 5-10 minutes, during which 4 μl ofprimer mixture were dispensed to the inside of each test tube cap, forthe hanging-drop hot start. Eva Green dye (final 1 μl of 20×; LifeTechnologies) was included in the 21 μl or added to the primer mix.After a further 10 minutes at room temperature, reactions were allowedto equilibrate in the hot block of a thermal cycler at 73° C. for 30seconds to inactivate DNase.

Hanging Drop Hot Start: The cycler program was then paused. Within aminute, each tube or 8-mer of tubes was given a brisk downward shake toadd the primers to the reaction, another downward shake, and returned to68° C. If electrophoresis was used, 1.7 μl was loaded onto 1.2% agarosegels. Control reactions showed that DNAse I is inactivated in 30 secondsif kept warm thereafter, yet 10-15 seconds of DNase at room-temperaturein contact with the primers was sufficient to prevent the LAMP reaction.MS2 RNA was diluted to 2-3 ng/μl in 0.1×LAMP reaction buffer, and addedto reactions as 1 μl.

RNase I Treatment.

RNase I treatment, when used, was carried out in 0.1-strength RT-LAMPreaction buffer, at 2 or 3 ng MS2 RNA per μl in a volume of 25 μl, withor without 0.2 units of E. coli RNase I (Epicentre). Incubation was atambient temperature for 10 minutes, before addition of LAMP reaction mixcontaining DNAse I as described above. 1 μl (2-3 ng RNA) was thusincluded in 26 μl reactions.

Results.

RT-LAMP with full-length Taq-D732N and 5′-exo mutations D119A and D119Nshowed improved clarity (see e.g., FIG. 19 ). Improved clarity of thebanding pattern, as usually observed for standard LAMP using Bst DNApolymerase, is apparent if an additional mutation near the metal-bindingsite of the 5′-exonuclease a.k.a. 5′-flap endonuclease is employed (seee.g., FIG. 19 ). D142A failed to have this effect. First column isD119D, i.e. wild-type at codon 119. All lanes used D732N Taq.

Example 3

The following example demonstrates strand displacement activity of fulllength mutant polymerase A111 (SEQ ID NO: 3). Methods are according toExample 2 unless otherwise described.

Mutant polymerase A111 (SEQ ID NO: 3) was originally identified asresistant to inhibitors of PCR assays of food (see U.S. PatentApplication Publication No. 2014/0113299, published 24 Apr. 2014). Froma mutagenized library, real-time PCR analysis identified enzyme variantsthat could still catalyze PCR in the presence of inhibitors. Clone A111was identified from assays with cracked pepper inhibitor (see e.g., FIG.5 ) and subsequent DNA sequencing (SEQ ID NO: 3) showed only a singlechange, D732N, from wild type Taq (SEQ ID NO: 1).

Strand-displacement activity (as in Bst polymerase) was attempted to beengineered into Klentaq1 (SEQ ID NO: 2) without compromising itsthermostability, by swapping sections of these two aligned sequences, orby inserting a DNA binding domain between K738 and 5739. Such effortswere not successful due to loss of thermostability sufficient for PCR.

Strand Displacement Activity

Mutant polymerase A111 (SEQ ID NO: 3) was tested in astrand-displacement half-dumbbell test (see e.g., FIG. 6 ). This assaytested whether an enzyme could displace 1.2 kb, go around a loop, andcome back to an end, producing a predicted, discrete 2.4 kb band. Asshown in FIG. 6 , mutant A111, i.e., TaqD732N (SEQ ID NO: 3) didsurprisingly well in this assay. This assay was designed so thatstrand-displacement would cause a band at 2.4 kb, rather than a smearwith other assays. But in a full length Taq mutant, the 5′-flap wouldchew away displaced strand, so a D732N mutation in Klentaq1 (truncatedA111, or KT-A111) (SEQ ID NO: 4) was studied.

Reverse Transcriptase Activity.

Another goal was to combine inhibitor-resistant mutations of Taq orKlentaq1 with significant reverse transcriptase (RT)-active mutationsdifferent than four already described (see e.g., Blotter et al. 2013Angewandte Chemie International Edition 52, 11935-11939; Ong et al. 2006Journal of Molecular Biology 361, 537-550).

Surprisingly, it was discovered that mutant polymerase A111 i.e.,TaqD732N (SEQ ID NO: 3) showed some RT-PCR activity (FIG. 20 ). Two MS2phage RNA targets, E and L, 210 bp each (MS2-E-FOR:AGGGTGCATATGAGATGCTTAC (SEQ ID NO: 24), MS2-E-REV:AGATACCTAGAGACGACAACCA (SEQ ID NO: 25), MS2-L-FOR: GATCGTATCCGCTCACACTAC(SEQ ID NO: 26), MS2-L-REV: TGCAATCTCACTGGGACATATAA (SEQ ID NO: 27),were amplified from 4 pg phage RNA with 0.25, 0.125 and 0.06 μl D732Nmutant enzyme, or 0.5, 0.25 and 0.125 μl wild-type Taq (NEB) (lanes 1-3each enzyme) in 35 μl reactions for 32 PCR cycles. Control reactions(lanes 4-6) contained no RNA. An RT-step of 30 min at 68° C. precededthe PCR amplification. 10 μl of amplified products were run in a 2.5%agarose gel, stained with ethidium bromide, along with a 100 bp DNAladder (lanes M). Reaction buffer was (1×) 50 mM Tris-HCl, pH 9.1, 16 mMAmSO₄, 2.5 mM MgCl₂, 0.025% Brij-58, 200 μM each dNTP. Each 35 μlreaction was also supplemented with 1/10 volume of PCR Enhancer Cocktail1 (DNA Polymerase Technology). RT+Cycling conditions (one-tube RT-PCR):RT step 2 min/75° C., 2 min/54° C., 30 min/68° C., PCR cycling 2 min/95°C. followed by 32 cycles 25 sec/95° C., 40 sec/54° C., 1 min/70° C.

This observation prompted generation of a Klentaq1-D732N enzyme (SEQ IDNO: 4) for LAMP with RNA template (RT-LAMP).

TABLE 1 Polymerase Mutations Klentaq1 mutant Codon 732 change 738,739insert Codons 742,743 KT732N D732N NONE EA wild-type KTflnC4RR D732NFLNCCPGCC RR KT-NRR D732N NONE RR KT-DRR NONE NONE RR

Mutant polymerase was tested in a 4 primer (non-optimized assay; 14.163LAMP F3 lambda GTAAAAACACCTCACGAGTT (SEQ ID NO: 28), 14.164 LAMP B3lambda TTTACGAACATTAAGCGACTT (SEQ ID NO: 29), 14.165 FIP LAMP lambdaTCCTACGGTCAAGAGAAGCAATAAA (−) CACCTAAGTTCTCACCGAAT (SEQ ID NO: 30),14.166 BIP LAMP CTTTCCACATGCAGGATTTTGG (−) ATGCACGCAATGGTGTAG (SEQ IDNO: 31)) for strand displacement activity run at 60° C. and 70° C.Positive control was Manta polymerase (a Bst DNA large fragmentpolymerase). Mutant polymerase KTflnC4RR (truncated polymerase havingD732N; FLNCCPGCC (SEQ ID NO: 32) insert at 738,739; and E742R and A743R(EA742RR)) showed strand displacement activity, notably at 70° C. whereManta was inactivated (see e.g., FIG. 9 ). KTflnC4RR was found tocatalyze LAMP for a lambda DNA target as well as Bst large fragment,after 3 hours.

TaqLamp1 (also named KTflnC4) differs in several ways from wild-type TaqDNA polymerase. 1) The first 278 amino acids are deleted, making itKlentaq1; 2) D732N; 3) E742R and A743R (EA742RR); 4) insertion, between738 and 739, of FLNCCPGCC.

After elimination all of the other changes, it was shown that D732Ncatalyzes LAMP. Additional mutant polymerases were tested in RT-LAMPreal-time traces using Eva Green indicator. Template was 3 ng of MS2 RNAin 26 μl reactions volumes. On the trace diagrams, inflected curvesindicate positive LAMP reactions (see, e.g., FIG. 14A). Curves or flattraces resulted for no-RNA and RNA+RNase (lines having no large circledata points), and their melt-curves showed no product melting near 86°C. (see, e.g., FIG. 14A). Although KT-RR (i.e., KT having both E742R andA743R substitutions, SEQ ID NO: 6) showed the fastest signal, it alsohad the highest NTC curve (no-template control) and RNase I curves;these negative control products melted at 80° C. or 65° C. As shown inFIG. 14B, genuine LAMP products melted at 85-86.5° C. Parallelexperiments without Eva Green usually have LAMP product (DNA ladder ongels) 5-10 minutes sooner than the inflection points shown here.

Control experiments showed that a modified Bst DNA polymerasecommercially provided for LAMP, can by itself surprisingly carry outRT-LAMP if MS2 RNA template is supplied without RNase I treatment.Additionally, studies of Bst 1.0 polymerase (New England Biolabs),without any RT (reverse transcriptase) enzyme has been shown to catalyzeRT-LAMP using the conditions described herein (see, e.g., lanes 5-8 ofFIG. 21 ).

It was shown that a single mutation to Taq DNA polymerase, D732N,confers a significant level of reverse transcriptase activity andconcomitant strand displacement ability. The ability of slightly mutatedTaq (only 1 codon) or Klentaq1 (1 or 2 codons) to catalyze LAMP wassurprising enough, but for reverse transcriptase activity to be aside-effect was even more surprising. Additionally, the E742R and A743R(EA742RR) mutations and the insertion mutation at position 738,739 showsurprising and unexpected results as well. It was found that Klentaq742RR (i.e., Klentaq having E742R and A743R substitutions) has RT-LAMPability, although it exhibits a high, competing level of primer-dimerformation compared to Taq D732N. Also, it was observed that Bst DNApolymerase has a high and useful level of RT activity, so that it cancarry out RT-LAMP in the PCR buffer that was used in these examples.

REFERENCES

-   1. Li, Y., Korolev, S. and Waksman, G. (1998) Crystal structures of    open and closed forms of binary and ternary complexes of the large    fragment of Thermus aquaticus DNA polymerase I: structural basis for    nucleotide incorporation. The EMBO journal, 17, 7514-7525.-   2. Tomita, N., Mori, Y., Kanda, H. and Notomi, T. (2008)    Loop-mediated isothermal amplification (LAMP) of gene sequences and    simple visual detection of products. Nature protocols, 3, 877-882.-   3. Blotter, N., Bergen, K., Nolte, O., Welte, W., Diederichs, K.,    Mayer, J., Wieland, M. and Marx, A. (2013) Structure and Function of    an RNA-Reading Thermostable DNA Polymerase. Angewandte Chemie    International Edition, 52, 11935-11939.-   4. Ong, J. L., Loakes, D., Jaroslawski, S., Too, K. and    Holliger, P. (2006) Directed evolution of DNA polymerase, RNA    polymerase and reverse transcriptase activity in a single    polypeptide. Journal of molecular biology, 361, 537-550.-   5. Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with    high fidelity and high yield from lambda bacteriophage templates.    Proceedings of the National Academy of Sciences, 91, 2216-2220.-   6. Yamagami, T., Ishino, S., Kawarabayasi, Y. and Ishino, Y. (2014)    Mutant Taq DNA polymerases with improved elongation ability as a    useful reagent for genetic engineering. Frontiers in microbiology,    5.-   7. Ignatov, K., Miroshnikov, A. and Kramarov, V. (1998) Substitution    of Asn for Ser<sup>543</sup> in the large fragment of <i> Taq</i>    DNA polymerase increases the efficiency of synthesis of long DNA    molecules. FEBS letters, 425, 249-250.-   8. Martin, B. R., Giepmans, B. N., Adams, S. R. and    Tsien, R. Y. (2005) Mammalian cell-based optimization of the    biarsenical-binding tetracysteine motif for improved fluorescence    and affinity. Nature biotechnology, 23, 1308-1314.-   9. Myers, T. W. and Gelfand, D. H. (1991) Reverse transcription and    DNA amplification by a Thermus thermophilus DNA polymerase.    Biochemistry, 30, 7661-7666.-   10. Dieffenbach, C. W. and Dveksler, G. (1993) Setting up a PCR    laboratory. Genome Research, 3, S2-S7.-   11. Kermekchiev, M., Kirilova, L., Vail, E. and Barnes, W. (2009)    Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA    amplification from whole blood and crude soil samples. Nucleic Acids    Research, 37, e40.-   12. Chandler et al. 2014 Frontiers in Microbiol., 5

1. A method of synthesizing a complementary DNA (cDNA) in a reversetranscriptase reaction comprising: forming an assay mixture comprising(A) a sample comprising a target RNA, (B) a primer which anneals to thetarget RNA (C) a buffer, and (D) at least one mutant polymerasecomprising a polypeptide sequence having (i) at least 95% sequenceidentity to SEQ ID NO: 1 or SEQ ID NO: 2, (ii) reverse transcriptaseactivity and DNA polymerase activity, and (iii) a D732N substitutionaccording to the numbering of wild type Taq polymerase of SEQ ID NO: 1,wherein the assay mixture does not include a separate reversetranscriptase enzyme in an amount sufficient to generate cDNA from thetarget RNA or in an amount sufficient to initiate reverse transcriptionof the target RNA; and allowing reverse transcription of the target RNAto synthesize the cDNA in the assay mixture.
 2. (canceled)
 3. The methodof claim 1, wherein the assay mixture does not include Mn⁺⁺ ion.
 4. Themethod of claim 1, wherein the sample comprising a target RNA is notpurified prior to addition to the assay mixture. 5.-8. (canceled)
 9. Amethod of amplifying a target nucleic acid in a reverse transcriptaseloop-mediated isothermal amplification (RT-LAMP) comprising: forming anassay mixture comprising (A) a sample comprising the target nucleicacid, wherein the target nucleic acid is a target RNA, (B) four or sixLAMP primers, wherein at least one primer anneals to the target RNA andwherein the four to six LAMP primers anneal to the cDNA or DNAsynthesized therefrom, (C) a buffer, and (D) at least one mutantpolymerase comprising a polypeptide sequence having (a) at least 95%sequence identity to SEQ ID NO: 1 or SEQ ID NO:2, (b) DNA polymeraseactivity, reverse transcriptase activity, and strand displacementactivity, and (c) a D732N substitution according to the numbering ofwild type Taq polymerase of SEQ ID NO: 1, wherein the assay mixture doesnot include a separate reverse transcriptase enzyme in an amountsufficient to generate cDNA from the target RNA or in an amountsufficient to initiate reverse transcription of the target RNA; andamplifying the target nucleic acid in the assay mixture in RT-LAMP. 10.(canceled)
 11. The method of claim 9, wherein the assay mixture does notinclude Mn⁺⁺ ion.
 12. The method of claim 9, wherein the samplecomprising a target DNA is not purified prior to addition to the assaymixture.
 13. The method of claim 9, wherein the assay mixture comprisesan inhibitory substance in an amount sufficient to cause a wild type Taqpolymerase to fail to amplify the target nucleic acid in the RT-LAMP.14. The method of claim 13, wherein the inhibitory substance compriseswhole blood, a blood traction, chocolate, peanut buffer, milk, seafood,meat, egg, or a soil extract.
 15. The method of claim 9, wherein the atleast one mutant polymerase comprises a polypeptide sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 8, or SEQ ID NO: 9, or a variantthereof having at least 95% sequence identity thereto and (A) DNApolymerase activity, (B) strand displacement activity, and (C) reversetranscriptase activity.
 16. The method of claim 9, wherein the at leastone mutant polymerase further comprises at least one mutation selectedfrom the group consisting of L609P, E626K, V649I, I707L, E708K, E708L,E708N, E708Q, E708I, E708W, E708R, E708V, E708S, E404G, G418E, V453L,A454S, R487G, I528M, L533R, D551 G, D578E, I599V, L657Q, K738R, L781 I,and E818V according to the numbering of wild type Taq polymerase of SEQID NO:
 1. 17. The method of claim 9, wherein the at least one mutantpolymerase comprises a polypeptide sequence having at least 95% sequenceidentity to SEQ ID NO:2.
 18. The method of claim 17, wherein the atleast one mutant polymerase further comprises substitutions E742R andA743R.
 19. The method of claim 9, wherein the at least one mutantpolymerase further comprises at least one substitution selected from thegroup consisting of D119A, D119N, E742R, and A743R according to thenumbering of wild type Taq polymerase of SEQ ID NO:
 1. 20. (canceled)